Our laboratory has utilized a three-dimensional culture model to investigate the phenotypic effects of genes implicated in breast tumorigenesis on the biology of mammary epithelial cells. This model system has revealed interesting cell-biological behaviors that are not detectable in standard monolayer cultures and has provided important mechanistic insights into processes and pathways that appear to play important roles in tumor initiation and progression in the mammary gland (e.g, filling of the lumen, escape from growth arrest, loss of polarity, etc). In the studies proposed in this application, we will further expand the application of these models for studies of tumor pathogenesis by examining the fate of single mammary epithelial cells that carry genetic alterations associated with breast cancer initiation or progression (e.g. expression of oncogenes or downregulation of tumor suppresors). Since tumors evolve from clonal alterations in isolated, individual cells, rather than globally in all cells within such a tissue, the proposed experimental models will more closely mimic the natural events associated with spontaneous tumor initiation and progression, and allow us to examine the influence of normal cells and the architectural organization of mammary structures on expression of phenotypic changes provoked by oncogenic insults. We will use the well-characterized MCF-10A immortalized human mammary epithelial cell model as well as other in vitro and in vivo mammary epithelial cell models that we are currently developing. The studies will involve systematic comparisons of the fate of cells subjected to distinct oncogenic insults in proliferating versus growth-arrested structures, in single cells versus the total cell population, and in immortalized versus primary cell three- dimensional structures. Findings from the in vitro models will be evaluated in vivo using inducible expression of cDNAs or shRNAs in single cells within the mammary gland. These studies promise to provide important information on (i) the fate of cell clones carrying oncogenic alterations, (ii) the role of epithelial cells within the microenvironment on the outcome of oncogene insults, (iii) the mechanisms of escape from suppressive influences of the microenvironment, and (iv) requirements for conferring a competitive advantage to tumor cells within tissue-like structures.